Ethanol

Appl Microbiol Biotechnol (2006) 69: 627642
DOI 10.1007/s00253-005-0229-x
MINI- REVI EW
Yan Lin
.
Shuzo Tanaka
Ethanol fermentation from biomass resources: current state
and prospects
Received: 18 July 2005 / Revised: 21 October 2005 / Accepted: 22 October 2005 / Published online: 6 December 2005
# Springer-Verlag 2005
Abstract In recent years, growing attention has been de-
voted to the conversion of biomass into fuel ethanol, con-
sidered the cleanest liquid fuel alternative to fossil fuels.
Significant advances have been made towards the technol-
ogy of ethanol fermentation. This review provides practical
examples and gives a broad overview of the current status of
ethanol fermentation including biomass resources, micro-
organisms, and technology. Also, the promising prospects of
ethanol fermentation are especially introduced. The pros-
pects included are fermentation technology converting xy-
lose to ethanol, cellulase enzyme utilized in the hydrolysis
of lignocellulosic materials, immobilization of the microor-
ganism in large systems, simultaneous saccharification and
fermentation, and sugar conversion into ethanol.
Introduction
With the inevitable depletion of the worlds energy supply,
there has been an increasing worldwide interest in alter-
native sources of energy (Aristidou and Penttila 2000;
Jeffries and Jin 2000; John 2004; Kerr 1998; Wheals et al.
1999; Zaldivar et al. 2001). It is now understood that it is
important to use biomass energy as a means of providing
modern energy to the billions who lack it. It would com-
plement solar, wind, and other intermittent energy sources
in the renewable energy mix of the future. One of the most
immediate and important applications of biomass energy
systems could be in the fermentation of ethanol from
biomass.
Biomass is seen as an interesting energy source for
several reasons. The main reason is that bioenergy can con-
tribute to sustainable development (Van den Broek 2000;
Monique et al. 2003). Resources are often locally available,
and conversion into secondary energy carriers is feasible
without high capital investments. Moreover, biomass energy
can play an important role in reducing greenhouse gas
emissions; since CO
2
that arises from biomass wastes would
originally have been absorbed from the air, the use of
biomass for energy offsets fossil fuel greenhouse gas emis-
sions (Lynd 1996). Furthermore, since energy plantations
may also create new employment opportunities in rural
areas, it also contributes to the social aspect of sustainability.
In addition, application of agro-industrial residues in bio-
processes not only provides alternative substrates but also
helps solve their disposal problem. With the advent of
biotechnological innovations, mainly in the area of enzyme
and fermentation technology, many new avenues have
opened for their utilization.
Nearly all fuel ethanol is produced by fermentation of
corn glucose in the US or sucrose in Brazil (MacDonald
et al. 2001; Rosillo-Calle and Cortez 1998), but any country
with a significant agronomic-based economy can use
current technology for fuel ethanol fermentation. This is
possible because, during the last two decades, technology
for ethanol production from nonfood-plant sources has
been developed to the point at which large-scale production
will be a reality in the next few years. Therefore, agronomic
residues such as corn stover (corn cobs and stalks), sugar-
cane waste, wheat or rice straw, forestry, and paper mill
discards, the paper portion of municipal waste and ded-
icated energy cropscollectively termed biomasscan
be converted into fuel ethanol. In this field, although
bioethanol production has been greatly improved by new
technologies, there are still challenges that need further
investigations. A further understanding of the ethanol
fermentation needs to be reached.
This review will focus on the current status of ethanol
fermentation including biomass resources, microorgan-
isms, technology, the practical examples, and especially the
promising prospects of ethanol fermentation.
Y. Lin
.
S. Tanaka (*)
Asian Center for Environmental Research,
Meisei University,
Tokyo, Japan
e-mail: tanakash@es.meisei-u.ac.jp
Biomass resources
There are various forms of biomass resources in the world,
which can be grouped into four categories. Wood residues
are by far the largest current source of biomass for energy
production. It comes from the wood product industry which
includes paper mills, sawmills, and furniture manufacturing.
Municipal solid waste is the next largest, followed by
agriculture residues and dedicated energy crops. Among
these biomass resources including short-rotation woody
crops and herbaceous crops, primarily tall grasses, dedicated
energy crops seem to be the largest, most promising, future
resource of biomass. This is because of the ability to obtain
numerous harvests from a single planting, which signifi-
cantly reduces average annual costs for establishing and
managing energy crops, particularly in comparison to con-
ventional crops (Monique et al. 2003).
Fermentation processes from any material that contains
sugar could derive ethanol. The varied rawmaterials used in
the manufacture of ethanol via fermentation are conveniently
classified into three main types of raw materials: sugars,
starches, and cellulose materials. Sugars (from sugarcane,
sugar beets, molasses, and fruits) can be converted into
ethanol directly. Starches (from corn, cassava, potatoes, and
root crops) must first be hydrolyzed to fermentable sugars by
the action of enzymes from malt or molds. Cellulose (from
wood, agricultural residues, waste sulfite liquor from pulp,
and paper mills) must likewise be converted into sugars,
generally by the action of mineral acids. Once simple sugars
are formed, enzymes from microorganisms can readily
ferment them to ethanol.
The most widely used sugar for ethanol fermentation is
molasses which contains about 50 wt% of sugar and about
50 wt% of organic and inorganic compounds, including
water. It is a thick, dark-colored syrup produced during
refinement of sugar. Since molasses contains microorgan-
isms which can disturb the fermentation, the molasses is
taken first to the sterilizer and then to the fermentor. Then it
is diluted with water to the mass fraction of 1018% to
reduce its viscosity in the pipeline. In addition, a very high
concentration of sugar can give too much ethanol and re-
sults in a prolonged fermentation time and an incomplete
sugar conversion. After the pH of the mash is adjusted to
about 45 with mineral acid, it is inoculated with yeast or
bacteria, and the fermentation is carried out nonaseptically
at 2032C for about 13 days.
Most agricultural biomass containing starch can be used
as a potential substrate for the ethanol fermentation by
microbial processes. These substrates include corn (maize),
wheat, oats, rice, potato, and cassava. On a dry basis, corn,
wheat, sorghums (milo), and other grains contain around
6075% (wt/wt) of starch, hydrolyzable to hexose with a
significant weight increase (stoichiometrically the starch to
hexose ratio is 9:10), and these offer a good resource in
many fermentation processes (Jackman 1987).
Fermentation of starch is somewhat more complex than
fermentation of sugars because starch must first be con-
verted into sugar and then into ethanol. Starch is first
hydrolyzed by adding -amylase to avoid gelatinization,
then cooked at high temperature (140180C). Next, the
liquefied starch is hydrolyzed to glucose with glucoamy-
lase. The resulting dextrose is fermented to ethanol with the
aid of microorganisms producing CO
2
as a coproduct.
During the process currently employed for industrial-scale
ethanol fermentation from starchy materials, high-tempera-
ture cooking (140180C) is very effective for fermentation
of starchy materials because it raises starch saccharification
efficiency and achieves high levels of ethanol production
under complete sterilization of harmful microorganisms.
However, production costs are high due to the high energy
consumption in the cooking process and the addition of
large amounts of amylolytic enzymes. So processes to
reduce the high production costs are required. To resolve
these difficulties, noncooking and low-temperature cooking
fermentation systems have been developed (Matsumoto et
al. 1985).
Industrial ethanol production has been reported using
various starchy materials such as corn, wheat, starch and
potatoes, cassava root (Lindeman and Rocchiccioli 1979),
corn stover (Kadam and McMillan 2003; Wilke et al.
1981), and starch (Maisch et al. 1979). Among many
starchy materials, cassava starch is an inexpensive fer-
mentable source. It is a tropical root crop produced in more
than 80 countries (Sasson 1990). About 20% of the cassava
starch was incorporated into animal feed. A similar amount
was converted into starch for industrial use and another
portion used for human food in some developing countries.
The rest was lost since cassava is perishable after harvest.
Harnessing the lost portion in addition to gains from new
high-yielding varieties with outputs of 100 tons per hectare
could provide the fermentation industry with an abundance
of raw material (Anthony et al. 1996). Fresh cassava has a
very high starch content, up to 30%. The content of sucrose
is about 4%. Dried cassava has 80% fermentable substrate.
However, cassava waste processing is difficult because it
is high in toxic materials. The potential toxicity of cassava
is due to the presence of cyanogenic glycosides, linamarin,
and lotaustralin, which on hydrolysis yield hydrogen cya-
nide on its peel. Traditional methods of cooking like boil-
ing and decanting remove cyanoglycosides to a certain
extent, but even then a certain amount of residual toxicity
remains in it (Westley 1980). Moreover, since starch
particles in cassava are bigger and there are some branched
structures, more glucoamylase has to be added into the
reactor. Furthermore, the nitrogen content of the cassava is
very low, so during the fermentation, nutrient has to be
added into the reactor to maintain the normal growth of the
microorganisms.
Among the three main types of raw materials, cellulose
materials represent the most abundant global source of
biomass and have been largely unutilized. The global pro-
duction of plant biomass, of which over 90% is lignocellu-
lose, amounts to about 20010
9
tons per year, where about
82010
9
tons of the primary biomass remains potentially
accessible. However, the effective utilization of the ligno-
cellulosic feedstock is not always practical because of its
seasonal availability, scattered stations, and the high costs of
transportation and storage of such large amounts of organic
628
material (Polman 1994). Recently, the enzymatic hydrolysis
of biomass cellulose is considered to be the most promising
technology available (Ogier et al. 1999; Yu and Zhang
2004). However, despite the work done, the industrial scale-
up of this process appears to be still hindered by techno-
logical issues or by the lack of a biomass refinery approach
in which ethanol is one of several products. In fact, because
raw material cost comprises more than 20% of the pro-
duction cost (Brown et al. 2001; Kaylen et al. 2000; Zhuang
et al. 2001), the optimization of the cellulose conversion
should be accomplished by correct management and uti-
lization of all process streams. A consequence of this situ-
ation is that even limited government intervention is still
crucial to maintaining ongoing research.
Furthermore, lignocellulose is a more complex substrate
than starch. It is composed of a mixture of carbohydrate
polymers (cellulose and hemicellulose) and lignin. The
carbohydrate polymers are tightly bound to lignin mainly
by hydrogen bonds but also by some covalent bonds. The
biological process for converting the lignocellulose to fuel
ethanol requires the following: delignification to liberate
cellulose and hemicellulose from their complex with lignin,
depolymerization of the carbohydrate polymers to produce
free sugars, and fermentation of mixed hexose and pentose
sugars to produce ethanol. Among the key processes de-
scribed above, the delignification of lignocellulosic raw
materials is the rate-limiting and most difficult task to be
solved. Another problem is that the aqueous acid used to
hydrolyze the cellulose in wood to glucose and other
simple sugars destroys much of the sugars in the process.
Extensive research has been carried out in this field for
decades (Yu and Zhang 2004), and the first demonstration
plant using lignocellulosic feedstocks has been in operation
in Canada since April 2004 (Tampier et al. 2004). It is
expected that the cost of lignocellulosic ethanol can
undercut that of starch-based ethanol because low-value
agricultural residues can be used.
General process
Besides the initial removal of large and unsuitable items,
key components of an integrated residual waste treatment
system based on ethanol fermentation include recyclable
materials recovery and removal of contaminants via me-
chanical preprocessing, initial hydrolysis process (conver-
sion to simpler compounds), fermentation of organics,
postfermentation purification of ethanol (by distillation or
filtration), gasification of solid residuals to provide process
heat, and treatment and disposal of wastewater.
Nearly all of the ethanol fermentation technologies use an
initial tipping floor removal of large or unsuitable materials,
followed by mechanical preprocessing to remove recycla-
bles and contaminants, and shredding of the material. Then
the material is processed through vessels using various
systems for the purpose of hydrolysis (breaking down to
simpler compounds) of the material. Depending on the
technology, this may include high temperature, acid treat-
ment, and/or high pressure. Following the initial hydrolysis
phase, the slurried material is then fermented to produce
alcohol, which is then purified through distillation and/or
filtration to produce the desired fuel-grade quality ethanol.
When cellulose was used as the raw material, the
cellulase responsible for enzymatic hydrolysis of pretreated
cellulosic biomass is strongly inhibited by hydrolysis
products: glucose and short cellulose chains. One way to
overcome cellulase inhibition is to ferment the glucose to
ethanol as soon as it appears in solution. Simultaneous
saccharification and fermentation (SSF) combines enzy-
matic hydrolysis with ethanol fermentation to keep the
concentration of glucose low (as shown in Fig. 1). The
accumulation of ethanol in the fermentor does not inhibit
cellulase as much as high concentrations of glucose, so SSF
is a good strategy for increasing the overall rate of cellulose
to ethanol conversion. In comparison to the process where
these two stages are sequential, the SSF method enables
attainment of higher (up to 40%) yields of ethanol by
removing end-product inhibition, as well as by eliminating
the need for separate reactors for saccharification and
fermentation (Bollk et al. 2000; Hari et al. 2001; Stenberg
et al. 2000). Other advantages of this approach are a shorter
fermentation time and a reduced risk of contamination with
external microflora, due to the high temperature of the
process, the presence of ethanol in the reaction medium,
and the anaerobic conditions (Emert and Katzen 1980;
Wyman 1994).
In spite of the obvious advantages presented by the SSF,
it has some drawbacks. These lie mainly in different
temperature optima for hydrolysis (4550) and fermenta-
tion (2835) (Ballesteros et al. 2004; Jeffries and Jin 2000;
Jeffries and Shi 1999). Besides, ethanol itself and some
toxic substances arising from pretreatment of the lignocel-
lulose inhibit the action of fermenting microorganisms, as
well as the cellulose activity (Targonski and Achremowicz
1986; Yu and Zhang 2004). Achieving microorganism
enzyme compatibility becomes a major issue in the SSF,
since some compounds (e.g., proteolytic enzymes) that are
released on cell lysis or are secreted by a particular strain
can degrade the cellulases, alternately, components in the
enzyme preparation, and reduce microbial viability leading
to cell lysis. On the whole, several process parameters must
be optimized: substrate concentration, enzyme to substrate
ratio, dosage of the active components (-glucosidase to
glucanase ratio) in the enzymatic mixture, and yeast
concentration.
Size reduction
Dilute acid
pretreatment
Enzyme
production
Ethanol
recovery
Simultaneous
saccharification and
fermentation
Residual solids
processing
Fig. 1 Schematic diagram of the conversion of biomass feedstock
to ethanol fuel
629
Microorganisms related to ethanol fermentation
Ethanol fermentation is a biological process in which
organic material is converted by microorganisms to simpler
compounds, such as sugars. These fermentable compounds
are then fermented by microorganisms to produce ethanol
and CO
2
. During the whole process of ethanol fermenta-
tion, there are mainly two parts for microorganisms. One is
for the microorganisms which convert fermentable sub-
strates into ethanol, and the other is to produce the enzyme
to catalyze chemical reactions that hydrolyze the compli-
cate substrates into simpler compounds.
Microorganisms producing ethanol
Several reports and reviews have been published on
production of ethanol fermentation by microorganisms,
and several bacteria, yeasts, and fungi have been reportedly
used for the production of ethanol. Those microbes that are
capable of yielding ethanol as the major product are shown
in Tables 1 and 2.
As shown in Tables 1 and 2, there are some micro-
organisms which can accumulate high concentrations of
ethanol. Historically, the most commonly used microbe has
been yeast, among the yeasts, Saccharomyces cerevisiae,
which can produce ethanol to give concentration as high as
18% of the fermentation broth, is the preferred one for most
ethanol fermentation. This yeast can grow both on simple
sugars, such as glucose, and on the disaccharide sucrose.
Saccharomyces is also generally recognized as safe (GRAS)
as a food additive for human consumption and is therefore
ideal for producing alcoholic beverages and for leavening
bread.
As with many microorganisms, S. cerevisiae metabo-
lizes glucose by the EmbdenMeyerhof (EM) pathway.
Beside this, the EntnerDoudoroff (ED) pathway is an
additional means of glucose consumption in many bacteria,
such as Zymomonas. The high ethanol yield and productiv-
ity observed for Zymomonas are a consequence of its unique
physiology. Zymomonas is the only microorganism that
metabolizes glucose anaerobically using the ED pathway as
opposed to the EM or glycolytic pathway (Matthew et al.
2005). The ED pathway yields only half as much ATP per
mole of glucose as the EM pathway. As a consequence,
Zymomonas produces less biomass than yeast, and more
carbon is funneled to fermentation products. Also, as a
consequence of the low ATP yield, Zymomonas maintains a
high glucose flux through the ED pathway. All the enzymes
involved in fermentation are expressed constitutively, and
fermentation enzymes comprise as much as 50% of the
cells total protein (Sprenger 1996).
Zymomonas mobilis is an unusual Gram-negative micro-
organism that has several appealing properties as a bio-
catalyst for ethanol production. The microorganism has a
homoethanol fermentation pathway and tolerates up to
120 g/l ethanol. It has a higher ethanol yield (510% more
ethanol per fermented glucose) and has a much higher
specific ethanol productivity (2.5) than Saccharomyces
sp. (Sprenger 1996). Furthermore, Z. mobilis is GRAS and
has simple nutritional needs. It is so well suited for ethanol
production that in the 1970s and 1980s, some researchers
advocated it as superior to S. cerevisiae. Despite its
advantages as an ethanologen, Z. mobilis is not well suited
for all of the biomass resources conversion because it
ferments only glucose, fructose, and sucrose. Moreover, for
Z. mobilis on synthetic media containing either glucose,
fructose or sucrose, the specific rates of sugar uptake and
ethanol production are at a maximum when utilizing the
glucose medium. In addition, S. cerevisiae is still preferred
by the industry because of the yeast hardiness.
Engineering Escherichia coli is another valuable bacte-
rial resource for ethanol production. The construction of E.
coli strains to selectively produce ethanol (Millichip and
Doelle 1989) was one of the first successful applications of
metabolic engineering. E. coli has several advantages as a
biocatalyst for ethanol production, including the ability to
ferment a wide spectrum of sugars, no requirements for
complex growth factors, and prior industrial use (e.g., for
production of recombinant protein). The major disadvan-
tages associated with using E. coli cultures are a narrow
and neutral pH growth range (6.08.0), less hardy cultures
compared to yeast, and public perceptions regarding the
danger of E. coli strains. The lack of data on the use of
residual E. coli cell mass as an ingredient in animal feed is
also an obstacle to its application.
Cellulose-to-ethanol biotransformation can be conducted
by various anaerobic thermophilic bacteria, such as Clos-
tridium thermocellum (Ingram et al. 1987), as well as by
some filamentous fungi, including Monilia sp. (Saddler and
Chan 1982), Neurospora crassa (Gong et al. 1981), Neuros-
pora sp. (Yamauchi et al. 1989), Zygosaccharomyces rouxii
(Pastore et al. 1994), Aspergillus sp. (Sugawara et al. 1994),
Trichoderma viride (Ito et al. 1990), and Paecilomyces sp.
(Gervais and Sarrette 1990). However, studies on the fer-
mentation process utilizing these microorganisms have
shown this process to be very slow (312 days) with a
poor yield (0.860 g/l of ethanol), which most probably is
due to the low resistance of microorganisms to higher
concentrations of ethyl alcohol. Another disadvantage of this
process (particularly in the case of bacterial fermentation) is
the production of various by-products, primarily acetic and
lactic acids (Herrero and Gomez 1980; Wu et al. 1986).
Hydrolysis enzymes and the related microorganisms
In addition to polymeric carbohydrates, raw material for
ethanol fermentation contains varying amounts of poly-
phenolic lignin and other extractables. These compounds
are not directly fermentable by most yeasts, and they must
be pretreated to hydrolyze the complicate compounds to
simple sugars (Zertuche and Zall 1982). Development of an
ideal pretreatment process is difficult, given that biomass
includes such sources as hardwood and softwood trees,
agricultural residues such as corn stover and nonrecyclable
paper waste.
630
Table 1 Yeast species which produce ethanol as the main fermentation product
Strain-species Temperature
(C)
pH
value
Carbon source and
concentration (g/l)
Nitrogen source and
concentration (g/l)
Incubation
time (h)
Concentration of
ethanol produced
(g/l)
References
27817-
Saccharomyces
cerevisiae
30 5.5 Glucose (50200) Peptone (2) and ammonium
sulfate (4)
1894 5.191.8 Vallet et al.
1996
L-041-S.
cerevisiae
30 or 35 Sucrose (100) Urea (1) or ammonium sulfate
(12)
24 2550 Leticia et al.
1997
181-S.
cerevisiae
(aerobic)
27 6.0 Glucose (10) Peptone (5.0) 40160 Todor and
Tsonka 2002
UO-1-S.
cerevisiae
(aerobic)
30 5.0 Sucrose (20) Ammonium sulfate (1) 6096 Camacho-
Ruiz et al.
2003
V5-S. cerevisiae 24 Glucose (250) 36 Virginie et al.
2001
ATCC 24860-S.
cerevisiae
30 4.5 Molasses (1.65.0) Ammonium sulfate (0.722.0) 24 518.4 Ergun and
Mutlu 2000
Bakers yeast-S.
cerevisiae
30 4.5 Sugar (150300) 192 53 (max) Roukas 1996
Bakers yeast-S.
cerevisiae
28 5.0 Sucrose (220) Peptone(5) and ammonium
dihydrogen phosphate (1.5)
96 96.71 Caylak and
Vardar 1996
Fiso-S. cerevisiae 30 5.0 Galactose (20150) Peptone, ammonium sulfate
and casamino acid (10)
60 4.840 da Cruz et al.
2003
A3-S. cerevisiae 30 5.0 Galactose (20150) Peptone, ammonium sulfate
60 4.836.8 da Cruz et al.
2003
L52-S. cerevisiae 30 5.0 Galactose (20150) Peptone, ammonium sulfate
60 2.432.0 da Cruz et al.
2003
GCB-K5-S.
cerevisiae
30 6.0 Sucrose (30) Peptone (5) 72 27 Kiran et al.
2003
GCA-II-S.
cerevisiae
30 6.0 Sucrose (30) Peptone (5) 72 42 Kiran et al.
2003
KR
18
-S.
cerevisiae
30 6.0 Sucrose (30) Peptone (5) 72 22.5 Kiran et al.
2003
CMI237-S.
cerevisiae
30 4.5 Sugar (160) Ammonium sulfate (0.5) 30 70 (max) Navarro et al.
2000
2.399-S.
cerevisiae
30 5.5 Glucose (31.6) Urea (6.4) 30 13.7 (max) Yu and Zhang
2004
24860-S.
cerevisiae
Glucose (150) Ammonium dihydrogen
phosphate (2.25)
27 48 (max) Ghasem et al.
2004
27774-
Kluyveromyces
fragilis
sulfate (4)
1894 48.96 (max) Vallet et al.
1996
30017-K.fragilis 30 5.5 Glucose (20120) Peptone (2) and ammonium
sulfate (4)
1996
30016-Kluyvero-
myces marxianus
sulfate (4)
1996
30091-Candida
utilis
sulfate (4)
1996
ATCC-32691
Pachysolen
tannophilus
30 4.5 Glucose (025) and
xylose (025)
Peptone (3.6) and ammonium
sulfate (3)
100 7.8 (max) Sanchez et al.
1999
631
These diverse feedstocks have caused researchers to test
numerous pretreatment processes ranging from hot water
and steam explosion treatments, to alkaline and solvent
pretreatments, to many useful versions of acid pretreatment
(Kaar and Holtzapple 2000; Maiorella 1985; Sun and
Cheng 2002). However, they acknowledge that detoxifi-
cation of acid-hydrolyzed lignin and other extractables in
the sugar hydrolysate will present additional costs for the
total hydrolysis process, costs that could be avoided en-
tirely if a fully enzymatic process (yet to be developed) is
implemented instead.
Traditionally, starch was, and still is, hydrolyzed to low
molecular weight dextrins and glucose using acid, but
enzymes have several advantages. First, the specificity of
enzymes allows the production of sugar syrups with well-
defined physical and chemical properties. Second, the
milder enzymatic hydrolysis results in few side reactions
and less browning. Indeed, for the production of glucose
syrups from starch, enzymic hydrolysis is essential. A
summary of starch degrading enzymes is shown in Fig. 2
(Hsu 1996).
There have been several reports about yeasts that could
produce extracellular -amylase and glucoamylase. These
include Candida tsukubaensis CBS 6389 (Aktinson and
Mavituna 1991), Filobasisium capsuligenum (Aktinson
and Mavituna 1991), Lipomyces kononenkoae (de Mot
and Verachtert 1985), Lipomyces starkeyi (Spencer-
Martins and Van Uden 1979), Saccharomycopsis bispora
(formerly Endomycopsis bispora) (Kelly et al. 1985),
Saccharomycopsis capsularis, Saccharomycopsis fibuli-
gera (Ebertova 1966; Stepanov et al. 1975), Schwannio-
myces alluvius (Gasperik et al. 1985), Schwanniomyces
castelli (Simoes-Mendes 1984), and Trichosporon pullu-
lans (Silla et al. 1984).
In addition, for the production of cellulolytic enzymes to
be used in the hydrolysis, the lignocellulose-degrading
fungus Trichoderma reesei can be used (Sharma 2000).
This fungus is able to metabolize pentose and hexose
sugars and also oligomers, and it is insensitive to inhibitors
generated from the lignocellulosic material, because these
are normally present in its natural environment.
In this field, it was investigated whether the cellulolytic
fungus T. reesei could degrade inhibitory compounds
present in a hemicellulose hydrolysate obtained after steam
pretreatment of willow and thereby decrease its inhibitory
effect on the ethanolic fermentation by S. cerevisiae. It was
also investigated whether the inhibitor containing fraction
could be used as a carbon source for the production of high-
quality cellulolytic enzymes to be used in the hydrolysis.
Kinetic models
Generally, economic restrictions force industrial processes
to work in a very small range of operating conditions. For
some batch processes which have long operating times in
each cycle and depend strongly on the operating variables,
it is very important to define the optimum conditions to
achieve sufficient profitability. Kinetic models describing
the behavior of microbiological systems can be a highly
appreciated tool and can reduce tests to eliminate extreme
possibilities.
Various kinetic models have been proposed in the lit-
erature for freely suspended cells in either batch or con-
tinuous operation (Ramon-Portugal et al. 1997; Reynders
et al. 1996; Tan et al. 1996). Unstructured models give the
most fundamental observations concerning microbial met-
abolic processes and can be considered a good approxi-
Table 2 Bacterial species which produce ethanol as the main
fermentation product
Mesophilic
organisms
Mmol ethanol produced per
mmol glucose metabolized
References
Clostridium
sporogenes
up to 4.15
a
Miyamoto 1997
Clostridium
indoli
(pathogenic)
1.96
a
Miyamoto 1997
Clostridium
sphenoides
1.8
a
(1.8)
b
Miyamoto 1997
Clostridium
sordelli
(pathogenic)
1.7 Miyamoto 1997
Zymomonas
mobilis
(syn. Anaerobica)
1.9 Miyamoto 1997
Zymomonas
mobilis subsp.
pomaceas
1.7 Miyamoto 1997
Spirochaeta
aurantia
1.5 (0.8) Miyamoto 1997
Spirochaeta
stenostrepta
0.84 (1.46) Miyamoto 1997
Spirochaeta
litoralis
1.1 (1.4) Miyamoto 1997
Erwinia
amylovora
1.2 Miyamoto 1997
Escherichia
coli KO11
0.70.1 Dien et al. 2003;
Matthew et al.
2005
Escherichia
coli LY01
4050 g ethanol produced/l Dien et al. 2003
Leuconostoc
mesenteroides
1.1 Miyamoto 1997
Streptococcus
lactis
1.0 Miyamoto 1997
Klebsiella
oxytoca
0.940.98 Matthew et al.
2005
Klebsiella
aerogenes
24 g ethanol produced/l Ingram et al. 1998
Mucor sp. M105 Ingram et al. 1998
Fusarium sp. F5 Ingram et al. 1998
632
mation when the cell composition is time dependent or
when the substrate concentration is high compared to the
saturation constant (Sonnleitner et al. 1997). Control
models for routine operation of industrial fermentations
are often based on simple, unstructured models since the
process computer will adjust the model parameters based
on the response of the system to disturbances.
When cultured in glucose media, unstructured models
have been found effective for describing the exponential
phase of the batch fermentation kinetics of cell growth and
ethanol production for strains of Z. mobilis ZM4 and ATCC
10988 (Moser 1985). These models, incorporated with the
bottleneck model approach, provide a base for establishing
a structured model that can describe the transient behavior
of a batch fermentation. An additional parameter, reflecting
the quality of the inoculum, is adjusted to match the model
prediction with the corresponding experimental result. In
continuous culture, the experimental findings suggest that
the specific substrate uptake rate is not linearly dependent
upon the specific growth rate, . A structured two-com-
partment model was introduced by Jobses et al. (1985) to
describe the fermentation of Z. mobilis. According to this
model, the specific substrate (glucose) uptake rate in
steady-state continuous culture is a nonlinear second-order
function of .
Gulnur et al. (1998) investigated the mathematical
description concerned with the basic metabolic processes
of S. cerevisiae in immobilized form. Glucose utilization,
ethanol production, and growth pattern of yeast cells
immobilized in calcium alginate gel beads were determined
in a stirred batch system using four different initial sub-
strate concentrations. Eleven different mathematical mod-
els taking into account the possibility of glucose or ethanol
inhibition on both yeast cell growth and ethanol production
were studied. The batch performance curves predicted by
the models were compared with the experimental data, and
the results were analyzed in terms of the possible effects of
initial condition (Doruker et al. 1995).
During the simulation of batch alcoholic fermentation
with the different initial conditions employed 11 different
models: the models of Monod, Moser, and Teissier were
used to represent inhibition-free substrate limitation kinet-
ics; the models of Andrews and Noack, Aiba and Luong
include substrate inhibition effects, whereas the models of
Levenspiel, Aiba, Jerusalimsky, Ghose and Tyagi, and
Hinshelwood include product inhibition effects. The mod-
els proposed by Monod and Hinshelwood were found to be
more appropriate for describing the batch growth and
ethanol production of immobilized S. cerevisiae at low and
high initial glucose concentrations, respectively (Gulnur et
al. 1998).
Structured models describing culture kinetics are
important in the control of bioreactors, as they provide a
mathematical description of the mechanism of the process
which are required for optimization and control. The ob-
jective of structured modeling is to obtain expressions that
quantitatively describe the behavior of the process under
consideration. A wide variety of models have been pro-
posed for the kinetics of the process; these range from very
simple models (Mori et al. 1970; Namba et al. 1984) to
more complex global models (Park and Toda 1990; Park et
al. 1990, 1991), which take into account the activating and
inhibiting effects of the substrate (glucose and oxygen) and
the product (ethanol and acetic acid; Oh et al. 2000).
However, none of these studies have put forward a general
model sufficiently well developed to permit the design of a
good simulator which is capable of performing simulations
with batch processes.
Moreover, structured models have been used to predict
the influence of operating parameters on cell concentration,
substrate utilization rate, and ethanol production rate.
These models may lead to the development of better strat-
egies for the optimization of the fermentation process to
ensure its economic viability. Although four factors (sub-
strate limitation, substrate inhibition, product inhibition,
and cell death) are known to affect ethanol fermentation,
none of these models accounts for these kinetic factors
simultaneously. Monods (1950) equation accounts only
for substrate limitation. The models of Hinshelwood (1946),
Holzberg et al. (1967), Egamberdiev and Jerusalimsky
(1968), Nagatani et al. (1968), Ghose and Tyagi (1979),
Hoppe and Hansford (1982), and Lee (1988) account only
for ethanol inhibition. The models of Aiba et al. (1968),
Aiba and Shoda (1969), and Luong (1985) include only
substrate limitation and substrate inhibition terms. An
appropriate ethanol fermentation model should therefore
account for the four kinetic factors.
A developed mathematical model capable of predicting
the cell, substrate, and ethanol concentrations during the
continuous anaerobic fermentation is necessary. However,
Starch-degrading
enzymes
-1,4-Glucanases
-1,6-Glucanases
Endo--1,4-glucanase
Exo--1,4-glucanases
Endo--1,6-glucanases
Exo--1,6-glucanase
Exomaltohexahydrolase
Exomaltotetrahydrolase
-Amylase
Glucoamylase
Isopullulanase
Pullulanase
Isoamylase
Exopullulanase

-Amylase
Fig. 2 A summary of starch
degrading enzymes
633
it cannot be expected that any kinetic model will be directly
applicable to a real process situation. Therefore, mathemat-
ical modeling should start with the simplest type, but it
must be reiterated, modified, and extended until it even-
tually leads to an adequate process kinetic model.
Pilot plants producing ethanol
Approximately 80% of the ethanol produced in the world is
still obtained from fermentations; the remainder comes
largely by synthesis from the petroleum product, ethylene.
The alcohol produced in the US is primarily used in
alcoholic beverages, but this is not always the case else-
where in the world. Brazil has embarked on a major
program to produce ethanol for fuel and thereby diminish
petroleum imports. As of 1984, approximately 7.9 million
tons of ethanol was produced by fermentation in Brazil,
with sucrose from sugarcane as the carbon source. The US
is also substantially increasing its fuel alcohol production,
originally because of the rapid increase in petroleum costs
during the 1970s, and the subsequent need for developing
alternative energy sources.
In spite of extensive research on fuel ethanol production
from biomass (shown in Table 3), until 1995, not a single
plant capable of converting cellulosic feedstock to ethanol,
via biological processing on the industrial scale, has been
put into operation anywhere in the world, although some
pilot scale plants have been commissioned (Szczodrak and
Fiedurek 1996).
During World War II, when wartime conditions changed
economic conditions and priorities, several ethanol-from-
cellulose (EFC) plants were built and operated in various
countries to provide an alternative fuel source. These
countries include Germany, Russia, China, Korea, Switzer-
land, and the US among others. Since the end of the war,
competition from synthetically produced ethanol has forced
many of these plants to close (Badger 2002). Since April
2004, the first demonstration plant using lignocellulosic
feedstocks in Canada has been in operation (Tampier et al.
2004). The target volume of 100 million liters of ethanol,
anticipated by 2006, will likely be met or exceeded by 2007.
There is also progress on pretreatment of softwood residues
and pentose fermentation (Natural Resources Canadas
management team 2005).
Currently, some countries in locations with higher eth-
anol and fuel prices are producing ethanol from cellulosic
feedstocks. It is only recently that cost-effective technol-
ogies for producing EFC in the US have started to emerge
(Badger 2002). In Canada, Iogen Corporation built a small
commercial-scale celluloseethanol plant using proprietary
enzymatic hydrolysis technology. In 1997, they partnered
with Petro-Canada to produce celluloseethanol beginning
with a 1-million-gallon-per-year ethanol demonstration fa-
cility, located at Iogens headquarters in Ottawa, using corn
stover and switchgrass (Energy & Environmental Research
Center, 2001). In summer 2005, a Swedish plant in
rnskldsvik started to produce ethanol from sawdust.
The production is still in a start-up phase, but the optimismis
high. In a not so distant future, Sweden could become self-
sufficient of ethanol from wood and wood residues, which
would be a much more sustainable way of supplying
ethanol to the Swedish market (Advanced course in LCA
2005).
Nowadays, in the field of sugar and starch utilization, the
large-scale application of modern bioenergy conversion
technologies has already occurred in a number of countries,
both in the industrialized and developing worlds. In the US,
the Minnesota Pollution Control Agency (MPCA) has
scheduled a public information meeting in early 2005 to
discuss the proposed Heron Lake BioEnergy ethanol proj-
ect. The proposed plant would cover 37 acres at a site about
1 mile northeast of the city of Heron Lake in Jackson
County. It would process 21.7 million bushels of corn
annually to produce 55 million gallons of ethanol and
193,300 tons of distiller dried grains (Sullivan 2005).
Another example is that of Brazil, a country that has com-
mitted itself to the development of its modern bioenergy
Table 3 The lists of pilot plants for ethanol production from biomass
Year Place Substrates Capacity
(ton/day)
Production
(l ethanol/day)
References
1976 US 1 Emert and Katzen 1980; Emert et al. 1980
1981 Canada Grain 960 27,400220,000 Robert 2004
1983 Cellulose 2,000 57,750 Emert et al. 1983
1983 Japan 720 150200 Morikawa et al. 1985a,b;
Morikawa and Tadokoro 1987
1984 Canada 1 Bente 1984
1988 France Cellulose 96 160190 kg/1,000 kg wood Ballerini et al. 1994; Nativel et al. 1992
1993 US Concentrated sweet whey 7.5 5,178 National Renewable Energy Laboratory 1996
2001 US Corn 155,000 Gary 2002
2002 US MSW 10,360,000 Badger 2002
2003 Canada Lignocellulosic 41,500 Tembec 2003
2005 US 570,000 MN Pollution Control Agency 2005
634
potential. Its sugarcane-based ethanol industry annually
produces around 15 billion liters from about 350 distilleries
and satisfies over 33% of the countrys gasoline needs
(Agama Energy 2003).
For the Global ethanol market, Brazil has more than 300
plants, producing 15 billion liters per year and supplying 3
million cars with pure ethanol. In the US, there are more
than 80 plants producing 10 billion liters per year, which it
intends to increase to 19 billion liters by 2010. China could
create 3 billion liters of ethanol per year. Indias annual
production of ethanol is 2.7 billion liters, and Eastern
Europes 2.5 billion liters. Western Europes production
ability is 2 billion liters and in Canada, 0.24 billion liters
could be achieved and possibly expanded to 1.4 billion
liters (Klein 2005).
Moreover, a fuel tax exemption is necessary for ethanol
to compete with gasoline. Biodiesel from waste vegetable
oil is already nearly competitive with conventional diesel,
which cannot be said of biodiesel made from far more
expensive virgin oils. It is foreseen that within the next 5
10 years, renewable, alternative transportation fuels from
biomass and wastes will be competitive with fuels derived
from petroleum at about US $ 0.2 per liter.
Generally, economic restrictions force industrial pro-
cesses to work in a very small range of operating conditions.
For some batch processes which have long operating times
in each cycle and depend strongly on the operating
variables, it is very important to define the optimum con-
ditions to achieve sufficient profitability. Kinetic models
describing the behavior of microbiological systems can be a
highly appreciated tool and can reduce tests to eliminate
extreme possibilities.
Most promising prospects
Ethanol fermentation involves significantly greater chal-
lenges, owing to the necessity of converting xylose as well
as glucose to ethanol in the process, the microorganism
enzyme compatibility in SSF, and the low rates of cellulose
hydrolysis. Recently, research has concentrated on the
development of improved processes; however, there are
still challenges that need further investigations.
Fermentation technology converting xylose to ethanol
Major fermentable sugars in hydrolyzate from cellulose
and hemicellulose are glucose and xylose. Glucose fer-
mentation to ethanol can be carried out efficiently by S.
cerevisiae. In contrast, xylose fermentation is challenging
because only a few traditional ethanol-producing micro-
organisms can readily ferment xylose, though many micro-
organisms utilize xylose as a carbon source. Efforts were
made to improve ethanol fermentation from xylose (Jeffries
and Shi 1999; Ho et al. 1999; Ingram et al. 1987; Zhang et
al. 1995).
However, low ethanol yields, by-product formation,
neutral pH requirement for growth, and intolerance to high
ethanol concentration are disadvantages in using bacteria in
large-scale fermentation (Bothast et al. 1999). Currently, the
bacterial conversion of xylose to ethanol has been studied
mostly with utilizing the recombinant microorganisms.
The recombinant E. coli was used for ethanol production
from xylose, and this ethanologenic strain (KO11) was able
to convert glucose and xylose to ethanol at yields of 103
106% of theoretical value (Gonzalez et al. 2003; Tao et al.
2001). The extra ethanol was thought to arise from fer-
mentation of carbohydrates present in the rich medium that
was not accounted for in the sugar balance. Moreover,
KO11 grows faster on xylose-containing medium than its
parent strain ATCC11303. Comparison of global gene
expression by microarray technology demonstrated that
KO11 overexpresses xylose metabolism genes (Tao et al.
2001). During the combination, two genes are needed, one
for pyruvate decarboxylase and another for alcohol dehy-
drogenase. These enzymes working together in the cell will
divert pyruvate away from other fermentation products to
ethanol. This would convert E. coli into an ethanologenic
microorganism. The steps by initial E. coli and ethanol-
ogenic E. coli in alcoholic fermentation are shown in Fig. 3
(Gottschalk 1986; Matthew et al. 2005).
Similarly, the ethanol-producing bacterium Z. mobilis
was metabolically engineered to broaden its range of
fermentable substrates to include the pentose sugar xylose.
Two operons encoding xylose assimilation and pentose
phosphate pathway enzymes were constructed and trans-
formed into Z. mobilis. The recombinant efficiently
fermented both glucose and xylose, which is essential for
economical conversion of lignocellulosic biomass to etha-
nol (Ingram and Doran 1995; Lynd et al. 2002; McMillan
et al. 1999; Sun and Cheng 2002; Zhang et al. 1995).
Currently, bacteria modified by this approach must operate
at neutral pH where control of invasion by other organisms
is more difficult than at the more acidic pH levels typical of
most yeasts.
Moreover, Tolan and Finn (1987) transformed Klebsiella
planticola ATCC 33531 with multicopy plasmids contain-
ing the pdc gene inserted from Z. mobilis, and expression of
the gene markedly increased the yield of ethanol to 1.3 mol
per mole of xylose, or 25.1 g/l. Concurrently, there was
significant decrease in the yield of other organic by-
products (i.e., formate, acetate, lactate and butanediol).
There have also been several yeast strains which were
capable of fermenting xylose to produce ethanol in batch
culture. Fein et al. (1984) isolated seven strains which were
capable of fermenting xylose to produce ethanol from
crude wood hydrolyzate in batch culture. Xylitol was
found to be one of the major by-products, and the amount
of xylitol varied depending on the strain used. Candida
tropicalis showed the greatest potential for ethanol pro-
duction from xylose. The crude acid hydrolyzate was
inhibitory to all strains of yeast, even at dilute hydrolyzate
concentrations. Strain acclimatization and chemical pre-
treatment resulted in a marked increase in utilization of
substrates in acidic crude hydrolyzate. In an attempt to
develop a xylose-fermenting yeast for industrial ethanol
production, UV light-induced mutants of Pachysolen
635
tannophilus have been isolated, which can grow faster on
xylose. Several other yeast strains for xylose utilization
have been reported (Jeewon 1997).
On the other hand, S. cerevisiae traditionally has been
used for ethanol production, such as beer and wine fer-
mentation. This yeast does not exhibit many of the limi-
tations encountered with bacteria. However, S. cerevisiae is
not able to ferment xylose. Therefore, metabolic engineer-
ing of xylose fermentation in S. cerevisiae is an attractive
approach (Sonderegger and Sauer 2003).
Although some significant progress can be noted in this
field, there are still some problems which exist. One of
them is ethanol inhibition. Ethanol inhibition of yeasts and
other microorganisms has received much attention in
microbial conversion of xylose to ethanol (Ghasem et al.
2004; Jeewon 1997; Palmqvist and Hahn-Hagerdal 2000).
Xylose-fermenting yeasts do not grow under anoxic
conditions and do not ferment when fully aerobic. There-
fore, development of fermentation glucose and xylose
efficiently is required for large-scale industrial application.
Cellulase enzyme
Using lignocellulosic materials such as agricultural
residues, grasses, forestry wastes, and other low-cost bio-
mass can significantly reduce the cost of raw materials
(compared to corn) for ethanol production. A reduction of
the cost of ethanol production can be achieved by reducing
the cost of either the raw materials or the cellulase en-
zymes. It was predicted that the use of genetically engi-
neered raw materials with higher carbohydrate content
combined with the improvement of conversion technology
could reduce the cost of ethanol by US $0.11 per liter over
the next 10 years (Wooley et al. 1999).
Xylose metabolism employs pathways distinctly differ-
ent from those involved in the utilization of glucose. With
most yeast, xylose metabolism requires aerobic conditions
at which cellular respiration is promoted; however, xylose
is fermented to ethanol in poor yields and at low rates. To
get around this problem, it has been proposed that the
xylose fraction first be converted to readily fermentable
xylulose, i.e., enzyme-mediated fermentation of xylose to
ethanol using the bacterial enzyme, xylose isomerase.
Reducing the cost of cellulase enzyme production is a
key issue in the enzymatic hydrolysis of lignocellulosic
materials. Genetic techniques have been used to clone the
cellulase coding sequences into bacteria, yeasts, fungi, and
plants to create new cellulase production systems with
possible improvement of enzyme production and activity.
Riley et al. (1996) and Wood et al. (1997) reported the
expression of recombinant endoglucanase genes from
Erwinia chrysanthemi P86021 in E. coli KO11, and the
recombinant system produced 3,200 IU endoglucanase/l
fermentation broth (IU, international unit, defined as a
micromole of reducing sugar as glucose released per
minute using carboxymethyl cellulose as substrate). The
thermostable endoglucanase E1 from Acidothermus cellu-
lolyticus was expressed in Arabidopsis thaliana leaves
(Ziegler et al. 2000), potato (Dai et al. 2000), and tobacco
(Hooker et al. 2001).
Immobilization
As in the case of microalgae culture in open ponds,
microecological engineering techniques will need to be
developed to maintain such strains in large systems which
could be subject to invasion and contamination by po-
tentially much faster growing wild microbes. Such micro-
ecological techniques would relieve the constraints of
having to maximize the amounts and activities of the
enzymes used in this process and/or maintain strictly
aseptic conditions which are not economical. If intact mi-

1/2 Glucose
(100 mol glucose)
Phosphoenol
Pyruvate
Succinate
10.7
Pyruvate
Acetyl-CoA Formate
2.4
CO + H
88.0 75.0
Acetaldehyde
Acetate
36.5
Ethanol
49.8
Lactate
79.5
4[H]
CO
2
2[H]
2[H]
2[H]
Acetyl-P
2[H]
1/2 Glucose
(100 mol glucose)
Phosphoenol
Pyruvate
Succinate
0.4
Pyruvate
Acetyl-CoA Formate
CO + H
Acetaldehyde
Acetate
0.7
Ethanol
Lactate
5.7
4[H]
+CO
2[H]
2[H]
2[H]
Acetyl-P
PDC
Acetaldehyde Ethanol
206
2[H]
ADH
Pet operon
B
A
Fig. 3 a Typical fermentation products made by a K12 Escherichia
coli fermenting glucose. Products are in moles produced per 100 mol
fermented glucose (Dien et al. 2003; Gottschalk 1986) with 91% of
the carbon accounted for as fermentation products. b Transforming
E. coli with pet operon diverts almost all glucose to ethanol. This
strain (KO11) also carries a mutation that blocks succinate pro-
duction. Amount of each fermentation product is shown per 100 mol
glucose (Dien et al. 2003; Ohta et al. 1991). Moles of CO
2
produced
was not measured, but should be 206 mol based on ethanol
production
636
crobial cells are directly immobilized, the removal of
microorganisms from downstream product can be omitted,
and the loss of intracellular enzyme activity can be kept to a
minimum level (Najafpour 1990).
Use of biofilm reactors for ethanol production has been
investigated to improve economics and the performance of
fermentation processes (Vega et al. 1988). Immobilization
of microbial cells for fermentation has been developed to
eliminate inhibition caused by high concentration of
substrate and product and also to enhance productivity
and yield of ethanol. The work on ethanol production in an
immobilized cell reactor (ICR) showed that production of
ethanol using Z. mobilis was doubled (Ghasem et al. 2004;
Takamitsu et al. 1993). The immobilized recombinant Z.
mobilis was also successfully used with high concentra-
tions of 1215% sugar (Yamada et al. 2002).
Recently, immobilized biomass activity has been given
more attention since it has been acknowledged to play a
significant role in bioreactor performance (Gikas and
Livingston 1997; Yamada et al. 2002). Frequently, im-
mobilized cells are subjected to limitations in the supply of
nutrients to the cells. Thus, because of the presence of
heterogeneous materials such as immobilized cells, there is
no convective flow inside the beads and the cells can
receive nutrients only by diffusion (Riley et al. 1996).
Immobilization of cells to a solid matrix is an alternative
means of high biomass retention. The cells divide within
and on the core of the matrix (Senthuran et al. 1997).
Simultaneous saccharification and fermentation
Simultaneous saccharification and fermentation (SSF)
gives higher reported ethanol yields and requires lower
amounts of enzyme because end-product inhibition from
cellobiose and glucose formed during enzymatic hydroly-
sis is relieved by the yeast fermentation (Banat et al. 1998;
McMillan et al. 1999). However, it is not easy to meet all
the requirements of industry due to their low rates of
cellulose hydrolysis, which is the stage limiting the rate of
alcohol production. Another problem arises from the fact
that most microorganisms used for converting cellulosic
feedstock cannot utilize xylose, a hemicellulose hydrolysis
product. Moreover, SSF requires that enzyme and culture
conditions be compatible with respect to pH and temper-
ature. T. reesei cellulases, which constitute the most active
preparations, have optimal activity at pH 4.5 and 55C. For
Saccharomyces cultures, SSF are typically controlled at
pH 4.5 and 37C.
To overcome the problems related to SSF, many species
of yeasts, as well as the bacterium Z. mobilis, have been
tested with cellulases produced by T. reesei mutants
(Chaudhuri and Sahai 1993; Haltrich et al. 1994; Spindler
et al. 1992). The currently most promising ethanologenic
bacteria for industrial exploitation are E. coli, Klebsiella
oxytoca, and Z.mobilis (Matthew et al. 2005). Genetic
engineering made it possible to transfer cellulose genes
from Trichoderma to S. cerevisiae (Shoemaker 1984).
However, the cellulases were produced at a concentration
too low to be useful. There is a group of microorganisms
(Clostridium, Cellulomonas, Trichoderma, Penicillium,
Neurospora, Fusarium, Aspergillus, etc.) showing a high
cellulolytic and hemicellulolytic activity, which are also
highly capable of fermenting monosaccharides to ethanol.
It may be possible, within this group of microorganisms, to
produce superstrains via genetic engineering capable of
hydrolyzing cellulose and xylan along with fermentation of
glucose and xylose to ethanol.
Moreover, to make the SSF process more effective, it has
also been found necessary to search for thermostable
strains capable of producing substantial amounts of ethyl
alcohol at temperatures optimal for saccharification and
suitably resistant to ethanol (Szczodrak and Targonski
1988).
Roychoudhury et al. (1992) have developed a notable
way of eliminating the negative effects which excessive
concentrations of ethanol have on yeast activity and
cellulased within the SSF system. They used a vacuum
cycling reactor where the concentration of ethanol was kept
at a relatively low level by its removal from the flash
chamber.
However, more efforts have to be made in the develop-
ment of microorganisms for industrial ethanol production.
In addition, it is important to keep the rate-limiting step in
mind. In SSF, the ethanol production rate is controlled by
the cellulase hydrolysis rate and not the glucose fermen-
tation, and hence, steps to increase the rate of hydrolysis
will lower the cost of ethanol production via SSF.
Sugar conversion
Since sugars are already available in a degradable form and
yeast cells can metabolize sugars directly, these substrates
require the least costly preparation. The other carbohy-
drates must be hydrolyzed to sugars before they can be
metabolized. Several studies have dealt with the economic
assessment of using cellulose hydrolysate, either from
waste (Cysewski and Wilke 1976; Green et al. 1989;
Maiorella et al. 1984; Wilke et al. 1976) or from wood
(Hinman et al. 1992; Marco et al. 2002). One disadvantage
with the application of these materials is their low sugar
content resulting in low cell and ethanol concentrations.
Hence, although starchy or cellulosic materials are cheaper
than sugar-containing raw materials, the requirement for
converting the starchy or cellulosic materials to ferment-
able sugars is a disadvantage of these substrates (Lynd et al.
2001).
Moreover, microorganisms used in industry are selected
to provide the best possible combination of characteristics
for the process and equipment being used. The selected
strains should have tolerance to high concentrations of
sugar and ethanol (Keim 1983; Oh et al. 2000).
Ethanol inhibition of yeasts and other microorganisms
has received much attention (Casey and Ingledew 1986) in
microbial production of ethanol. Lucas and van Uden
(1985) investigated the effects of temperature on ethanol
tolerance and thermal death of Candida shehatae and
637
determined that it was more tolerant of ethanol at lower
temperatures. Du Preez et al. (1987) quantitatively eval-
uated the effects of ethanol on the growth of the xylose-
fermenting yeasts C. shehatae and Pichia stipitis using
Luong kinetics. The effect of ethanol on metabolic rate has
been examined with ethanol added exogenously. Both
Lucas and van Uden (1985) and du Preez et al. (1987)
placed cells into media containing different concentrations
of ethanol and measured the specific growth rate which
ensued. Unfortunately, less inhibition is observed with
exogenous ethanol than with the same concentration of
ethanol produced endogenously (Hoppe and Hansford
1982; Novak et al. 1981; du Preez et al. 1987). Some have
claimed that the apparently greater inhibition by endoge-
nously produced ethanol reflects the tendency of actively
fermenting cells to accumulate ethanol intracellularly
(Casey and Ingledew 1986; Ghasem et al. 2004); however,
the yeast plasma membrane is known to be very permeable
to ethanol, which casts doubt on this hypothesis. Whatever
the reason for the different effects of externally added and
internally generated ethanol, realistic assessments of eth-
anol inhibition ought to involve ethanol generated in situ.
On the other hand, since the distillation cost per unit
amount of ethanol produced is substantially higher at low
ethanol concentrations (Zacchi and Axelsson 1989), sev-
eral investigators have dealt with the idea of concentrating
sugar solutions prior to fermentation (Cysewski et al. 1976;
Iraj et al. 2002; Oh et al. 2000; Zacchi and Axelsson 1989).
Clearly, it is necessary to solve the problem between the
concentration of ethanol produced and sugar added if an
economically sustainable system is to be created using this
method.
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Appl Microbiol Biotechnol (2006) 69: 627642

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